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/lp/springer-journals/effect-of-traditional-herbal-medicine-danggui-yukhwang-tang-on-post-VVUFBzVW4Z
- Publisher
- Springer Journals
- Copyright
- Copyright © The Author(s) 2023
- ISSN
- 2468-0834
- eISSN
- 2468-0842
- DOI
- 10.1186/s13765-022-00761-3
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- See Article on Publisher Site
Abstract
The decrease in estrogen due to menopause leads to impaired lipid metabolism and is closely related to the increase in metabolic syndrome due to weight gain. Hormone replacement therapy is effective for menopause, but with an increased risk of side effects. Danggui‑ yukhwang‑tang (DY T ) is a traditional drug, comprising seven herbs, used to treat diseases like slight fever with sweating, blood‑flow disorders, and neurasthenia. However, the effect on meno ‑ pausal obesity has not been reported. This study aimed to investigate the inhibitory effect of DY T on weight gain in female rats fed a high‑fat diet after ovariectomy. Adipocyte differentiation was effectively reduced by DY T in 3T3‑L1 cells, and the mRNAs of PPARγ, C/EBPα, and FABP4, which are adipogenesis‑related genes, were reduced. In the in vivo study, OVX and HFD elevated body weight; however, its induction significantly decreased in the DY T ‑treated groups. The serum lipid profile was also examined, and DY T treatment significantly decreased LDL ‑ cholesterol, triglyceride, and total cholesterol levels compared with the OVX and OVX + HFD groups. DY T treatment effectively reduced the temperature(s) of the tail and body in the rats. The study demonstrates that DY T inhibits adipogenic differentiation, hypercholesterolemia, and weight gain in a post‑menopausal rat model by regulating adipogenic markers (PPARγ, C/ EBPα, FABP4) and the serum lipid profile in OVX + HFD rats. Keywords Danggui‑ yukhwang‑tang (當歸六黃湯, Dāngguīliùhuángtāng), Menopausal syndrome, Adipogenic differentiation, Obesity symptoms such as vasomotor symptoms (hot flashes and Introduction night sweats), osteopenia, osteoporosis, mental disorders, The decline in ovarian activity due to natural aging and sexual dysfunction [4]. Hormonal decline is closely causes estradiol loss, clinically diagnosed as ‘menopause,’ associated with obesity, type 2 diabetes, and impaired when there is no menstruation for one year. It mainly lipid metabolism, which may contribute to weight gain occurs between the ages of 45 and 55, and the number and the pathogenesis of metabolic syndromes including of menopausal patients worldwide is expected to increase cardiovascular disease (CVD) [5, 6]. due to the extension of women’s life expectancy [1–3]. Proliferator-activated receptor γ (PPARγ) and Women diagnosed with menopause complain of various CCAAT/enhancer-binding protein α (C/EBPα) are key regulators of adipocyte differentiation and lipid accu - Dong Ho Jung and Hyun Yang contributed equally to this work mulation. These adipogenic factors are directly impli - *Correspondence: cated in adipogenesis pathways in adipocytes, including Byoung‑Seob Ko fatty acid-binding protein 4 (FABP4) and fatty acid bsko@kiom.re.kr synthase (FAS) [7]. Several studies have reported that KM Convergence Research Division, Korea Institute of Oriental Medicine (KIOM), 1672 Yuseong‑daero, Yuseong‑Gu, Daejeon 34054, Republic the activity of the estrogen receptor α (ERα), a recep- of Korea tor for 17β-estradiol, decreases adipogenesis and lipid © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Jung et al. Applied Biological Chemistry (2023) 66:6 Page 2 of 12 accumulation through inhibition of PPARγ activity and related genes using the 3T3-L1 pre-adipocyte cell line. decreases the activity of lipolytic enzymes due to ERα- Furthermore, we investigated the efficacy of DYT in post- induced upregulation of α-adrenergic receptors (αAR) menopausal obesity and hot flashes in an ovariectomized [8, 9]. Therefore, 17β-estradiol (E ) deficiency upregu - (OVX) high-fat diet (HFD) animal model. lates the expression of adipogenic genes required for energy storage while downregulating β-oxidation- Materials and methods related genes for energy production as the body’s fuel DYT preparation source. This phenomenon causes negative fat and The seven herbal medicines that constitute DYT were energy metabolism changes, resulting in an imbalance purchased from Gwangmyeong-dang (Daejeon, Korea) between them, eventually leading to obesity [10]. In and approved by the botany specialist Dr. Byeong-Sub addition, hot flashes are also a symptom with a high Ko (Korean Institute of Oriental Medicine, KIOM). DYT incidence in menopausal women. A study on the asso- (26.2 g) was extracted by heating a mixture of seven ciation between hot flashes and obesity showed that herbal medicines (Rehmanniae Radix Preparat (24%), increased abdominal fat was associated with increased Astragali Radix (21%), Angelicae Gigantis Radix (17.6%), hot flashes [11, 12]. In addition, a study using ova- Scutellariae Radix (15.3%), Rehmanniae Radix (8.8%), riectomized (OVX) mice by Wang et al. confirmed an Phellodendri Cortex (7.3%), and Coptidis Rhizoma (6%)), increase in tail skin temperature, body weight, and in a tenfold volume of water at 70 °C for 3 h. The extract abdominal fat [13]. Therefore, these studies suggest that was filtered through 0.4 μm filter paper (Whatman Inter - alleviating hot flashes may be possible due to body fat national, Maidstone, UK), and the filtered extract was reduction. evaporated using a rotary vacuum evaporator (N-1200A; Hormone replacement therapy (HRT) was preferred Eyela, Tokyo, Japan) and then freeze-dried (yield 29.31% among various treatments because of its quick and w/w). DYT extract (KIOM-PH-130026) was stored at the immediate effect; however, after the Women’s Health Korea Institute of Oriental Medicine (KIOM, Daejeon, Initiative (WHI) announcement, the frequency of use Korea). decreased owing to various side effects and complica - tions [14, 15]. Therefore, many researchers are exploring Chromatographic conditions for HPLC alternative medicines and methods that can replace HRT The HPLC analysis was conducted with a Shimadzu LC- [16]. In Korea, the prescription danggui-yukhwang-tang 20A Prominence Series system (Shimadzu Corporation, (DYT) was widely used for fever, diabetes, perimeno- Kyoto, Japan) equipped with a quaternary pump (LC- pausal syndrome, psoriasis, tuberculosis, and hyperthy- 20AD), vacuum degasser (DGU-20A3R), auto-sampler roidism [18, 19]. In Chinese, DYT is called Dāngguī liù (SIL-20A), column oven (CTO-20A), and photodiode- huángtāng (当归六黄汤). DYT consists of seven medici- array detector (SPD-M20A). The chromatographic data nal herbs: Rehmanniae Radix Preparat (RRP), Astragali were interpreted using Lab-Solutions Multi-PDA soft- Radix (AR), Angelicae Gigantis Radix (AGR), Scutellariae ware. Chromatographic separation was performed on Radix (SR), Rehmanniae Radix (RR), Phellodendri Cor- an Atlantis C18 (4.6*250 nm, 5um, Phenomenex). The tex (PC), and Coptidis Rhizoma (CR). However, no stud- column oven was maintained at 30 °C, the detection ies have been conducted to determine whether DYT is was conducted at λ = 254 nm, and online UV absorption effective for treating menopausal obesity. Therefore, in spectra were recorded in the range of 190 to 400 nm. The this study, we investigated adipocyte differentiation and gradient solution system was used for HPLC analysis for Table 1 Linearity and contents (ug/mg) of DY T compounds a 2 b Compound name t (min) Equation (linear model) r Mean (μg/mg) 5‑HMF (1) 10.977 y = 74.905x—22.201 0.9994 0.8602 Nodakenin (2) 25.597 y = 20.545x—30.404 0.9991 2.9058 Baicalin (3) 30.601 y = 39.986x—41.79 0.9993 5.7878 Berberine (4) 36.097 y = 41.842x—76.286 0.9990 1.1507 Baicalein (5) 38.09 y = 63.327x – 156.2 0.9997 ND Formononetin (6) 40.03 y = 63.853x – 9.8768 0.9988 ND Wogonin (7) 43.29 y = 82,563x – 65.128 0.999 ND Decrusin (8) 51.215 y = 142.02x—0.1912 0.9993 0.0168 a y: peak area at 254 nm; x: standard concentration (mg/mL). b r : coefficient of determination with 8 indicated points on the calibration curves Jung et al. Applied Biological Chemistry (2023) 66:6 Page 3 of 12 standard compound detection, and the analysis condi- an EZ-CyTox cell viability assay kit (Daeil Lab Service, tions are shown in Table 1. Flow rate was 1.0 mL/min and Seoul, Korea). The absorbance was measured at 450 nm the injection volume were 1 μL. The standard compounds after the cells were incubated with EZ-CyTox solution (10 were obtained from Sigma-Aldrich (St. Louis, MO, USA). µL/well) for 4 h using a microplate reader (SynergyTM The eight chemical compounds had purities of ≥ 95% HT; BioTek Instruments, Inc., Winooski, VT, USA). (Fig. 1a). The stock solution was prepared at concentra - tions of 1 mg/mL in 100% MeOH. The working solutions Mouse pre‑adipocytes (3T3‑L1) culture and adipocyte were serially diluted with methanol to obtain final con - differentiation centrations of 0.0001 mg/mL and they were stored at 4 °C 3T3-L1 pre-adipocytes were purchased from the Ameri- prior to analysis. can Type Culture Collection (ATCC, Rockville, MD, USA). Cells were maintained in DMEM containing 10% Cell viability assay newborn calf serum (NBCS) and 0.5% antibiotics (peni- After overnight incubation in a 96-well plate at 2 × 10 cillin 100 U/mL and streptomycin 100 μg/mL) medium cells/well, the 3T3-L1 pre-adipocyte cells were exposed (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) in to DYT (1, 10, 100, 200, 300, 400, 500, and 600 μg/mL) in a humidified atmosphere of 5% CO at 37 °C. For adipo- the medium for 24 h. Cell viability was determined using cyte differentiation, 3T3-L1 cells were incubated for two (a) 2 : Nodakenin 1 : 5-(Hydroxymethyl)-2-furaldehyde 3 : Baicalin 4 : Berberine 6 : Formononetin 7 : Wogonin 8 : Decrusin 5 : Baicalein (b) Fig. 1 The structures of eight compounds and HPLC analysis data of DY T extract. The HPLC chromatograms of the standard mixture 0.2 mg/mL, λ = 254 nm (b, top panel). The extracted DY T sample 1.0 mg/mL, λ = 254 nm (b, middle panel). The double check of nodakenin (2) and decrusin (8) from ARG was confirmed at 330 nm (b, lower panel) Jung et al. Applied Biological Chemistry (2023) 66:6 Page 4 of 12 Experimental animals and treatments days with 0.5 mM 3-isobutyl-1-ethylxanthine (IBMX), Female SD rats (5 weeks old) were purchased from 1 μM dexamethasone, 1.67 μg/mL insulin (Sigma Aldrich, Dooyeol Biotech Inc. (Seoul, Korea) and allowed to adapt St Louis, MO, USA) in phenol-free DMEM/F12 (1:1) to laboratory conditions (temperature, 20 ± 2 °C; relative medium containing 10% charcoal-stripped FBS (CH-FBS, humidity, 45 ± 5%; light/dark cycle, 12 h) for one week. Gibco). It was then incubated with 1.67 μg/mL insulin for After acclimatization at the experimental animal center of two days and then incubated in phenol-free DMEM con- the Korea Institute of Oriental Medicine, anesthesia was taining 10% CH-FBS for four more days to observe lipid induced using Avertin, and the skin was moved to the left accumulation. and right to remove the ovary (OVX). A post-menopausal model was established by inducing symptoms for eight Oil red O staining and total lipid droplets quantification weeks after surgery. The rats (n = 56) were divided into To measure total intracellular lipid content, 3T3-L1 cells eight groups (n = 7 sham, n = 7 OVX, n = 7 OVX + HFD, were stained by the Oil Red O (ORO) method as previ- n = 7 OVX + HFD + E , n = 7 OVX + HFD + SV, n = 7 ously described [20]. Briefly, after cell differentiation, OVX + HFD + DYT500, n = 7 OVX + HFD + DYT1000, cells were washed twice with phosphate-buffered saline and n = 7 OVX + HFD + DYT1500). After two weeks of (PBS) and fixed with 10% formalin for 30 min at room OVX, to assess the effects of DYT in preventing post- temperature. Cells were washed with 60% isopropanol menopausal symptoms, rats were fed the AIN-76A diet and stained with Oil Red O solution (Sigma-Aldrich, St as normal control (Sham), AIN-76A with 17β-estradiol Louis, MO, USA) for 1 h. After staining, the cells were (40 μg/kg/BW; Sigma-Aldrich, MO, USA) as a positive sequentially washed with 70% ethanol and PBS and visu- control I (OVX + HFD + E ), AIN-76A with simvastatin alized under an optical microscope (Olympus, Tokyo, (20 mg/kg BW; Sigma-Aldrich, MO, USA), and AIN- Japan). The stained lipid droplets were dissolved in 100% 76A supplemented with DYT (DYT500; 500 mg/kg/BW, isopropanol and quantified using a microplate reader DYT1000; 1000 mg/kg/BW or DYT1500; 1500 mg/kg/ (BioTek Instruments, Inc.) by measuring the optical BW) for 6 weeks. All animal experimental procedures absorbance at 500 nm. were approved by the Ethics Committee of the Korea Institute of Oriental Medicine (Approved No. 17–029; Quantitative real‐time polymerase chain reaction Daejon, South Korea). (qReal‑time PCR) Total RNA was extracted from 3T3‐L1 cells using the RNeasy Mini kit (QIAGEN Inc., Germantown, MD, Cholesterol and triglyceride measurements in vitro USA), and the RNA concentration was assessed using a and in vivo NanoDrop 2000 spectrophotometer (Thermo Scientific, The total triglyceride (TG) and total cholesterol (TC) Waltham, MA, USA). The extracted RNA was reverse- contents of 3T3-L1 cells were measured using TG and transcribed into cDNA using an iScript cDNA syn- TC quantification kits (ASAN Pharm, Co., Ltd, Seoul, thesis kit (Bio-Rad, Hercules, CA, USA). The mRNA Korea) [20]. Briefly, the treated cells were collected and expression levels were quantified by quantitative real- vortexed by adding chloroform: isopropanol: tween-20 time PCR using SYBER Green PCR Master Mix and a (7:11:0.1), and the extract was separated by centrifugation 7500 Real‐Time PCR system (Applied Biosystems, Foster at 15,000×g for 10 min. The supernatant was transferred City, CA, USA) according to the manufacturer’s proto- to a new tube and dried at 50 °C to completely remove col. The primers targeting adipogenic markers were as the residual chloroform. After sufficiently dissolving the follows: mouse C/EBPα sense, 5′-CGA CTT CTA CGA dried lipids in 150 μL of assay buffer, the TG and TC lev - GGT GGA GC-3′; mouse C/EBPα anti-sense, 5′-TCG els were measured using a microplate reader (BioTek ATG TAG GCG CTG ATG TC-3′; mouse PPARγ sense, Instruments, Inc.) at 510 and 500 nm. The results were 5′-GGA AGC CCT TTG GTG ACT TTA TGG-3′; normalized to quantitative protein values. mouse PPARγ anti-sense, 5′-GCA GCA GGT TGT CTT Blood samples were collected directly from the inferior GGA TGT C-3′; mouse FABP4 sense, 5′-AAT CAC CGC vena cava using a 1 mL syringe at the end of the experi- AGA CGA CAG-3′; mouse FABP4 anti-sense, 5′-ACG ment. Serum was obtained by centrifugation at 4000×g CCT TTC ATA ACA CAT TCC-3′; mouse GAPDH for 10 min and stored at −70 ℃ until use. Cholesterol sense, 5′-TTG ATG CGA ACA ATC TCC AC-3′; mouse (LDL-C, HDL-C, and TC) and TG concentrations were GAPDH anti-sense, 5′-CGT CCC GTA GAC AAA measured using an ELISA kit (BioVision Inc., Waltham, ATG GT-3′. Fold-changes are presented as 2 − ∆∆Ct MA, USA), following the manufacturer’s protocol. (∆∆Ct = ∆Ct control − ∆ Ct treatment). Briefly, 50 μL of serum collected from experimental ani - mal groups or standard and 100 μL of biotinylated anti- body cocktail was added to each well and allowed to react Jung et al. Applied Biological Chemistry (2023) 66:6 Page 5 of 12 for 2 h with 37 ℃. Subsequently, the wells were treated comparing and analyzing the maximum UV absorbance with 100 μL of HRP conjugate for 1 h, then reacted with profiles of each compound, and 254 nm was selected as 90 uL substrate for 15 min. Finally, 50 uL stop solution the optimal single wavelength at which all eight com- was treated and reacted, and then optic density was pounds could be detected. The double check of each measured with a microplate reader at 450 nm within compounds was confirmed at 254 nm, 330 nm. The linear 10 min. A standard curve of each cholesterol was pre- regression analysis of each compound with a correspond- pared and linear regression analysis was performed using ing standard was carried out, the results showed good PRISM software (GraphPad Inc., CA, USA) based on this linearity with r values exceeding until 0.9990 to 0.9999, standard curve. respectively. As a result, the three standard compounds selected, baicalein (5) and Formononetin (6), Wogonin Histological examinations of the liver and adipose tissue (7) were not detected in DYT. Under these HPLC condi- in ovariectomized high‑fat diet animal model tions, all compounds were free of interference from any Adipose tissues (peritoneal fat) were fixed with 10% other components and showed retention times of 10.97 neutral buffered formalin. These tissues were embedded (1), 25.75 (2), 30.60 (3), 36.09 (4), 38.09 (5), 40.03 (6), in paraffin, sliced into 5 μm-thick sections, and stained 43.29 (7), and 51.21 (8) min, respectively. The contents with H&E (Sigma Aldrich, St Louis, MO, USA). For the of the five compounds were successfully determined to Oil Red O staining of the liver tissue, OCT-embedded be 0.8602 μg/mg for 1, 2.9058 μg/mg for 2, 5.7878 μg/mg frozen tissues were sectioned at 10 μm, stained with for 3, 1.1507 μg/mg for 4, 0.0168 μg/mg for 8 (Fig. 1 and 0.5% Oil Red O, and counterstained with hematoxylin. Table 1). The cells were fixed in 4% paraformaldehyde solution for 10 min, rinsed with PBS, and stained with Oil Red O Eec ff t of DYT on 3T3‑L1 cell cytotoxicity for 30 min. All tissue samples were evaluated and pho- To investigate the cytotoxicity of DYT, 3T3-L1 cells were tographed under a light microscope in a blinded manner treated with various DYT concentrations (1, 10, 25, 50, (BX43; Olympus). Images were captured using an Olym- 75, 100, 200, 300, 400, 500, and 600 μg/mL) for 24 h. As pus DP-73 microscope (Olympus) and CellSens stand- shown in Fig. 2a, cytotoxicity by DYT was not observed ard software (Olympus). Adipose and liver tissue images at any treatment concentration or time compared with were analyzed using ImageJ software (ver. 1.51j8). the control group (untreated group). In contrast, sig- nificant cell proliferation was observed in the DYT 500 Tail temperature and infrared body temperature (p < 0.01) and 600 μg/mL (p < 0.001) treatment groups The tail temperature of the rats was measured by attach - for 24 h. Cytotoxicity was not observed at 600 μg/mL ing an infrared thermometer (Bioseb, Cheville, France; DYT, but it did not completely dissolve in water; hence, BIO-IRB153) to the tail. The surface temperature of the the final treatment concentration was determined to be rat and its immediate surroundings were recorded using 500 μg/mL. an infrared digital thermographic camera (IRIS-XP; Medicore, Gyunnggi-do, South Korea) placed 100 cm Inhibitory effect of DYT on lipid production above the rats. The rats were photographed in the same and accumulation in 3T3‑L1 cells gesture as possible in the recorded video. Based on our cell cytotoxicity results, we examined the anti-adipogenic effect of DYT in vitro using 3T3-L1 pre- Statistical analysis adipocyte cells (Fig. 2b). First, we induced 3T3-L1 cell Data are represented as the mean ± SD. Differences differentiation by treating the cells with E and DYT at between means were obtained by conducting one-way the indicated concentrations in differentiation medium ANOVA followed by Tukey’s multiple comparison test supplemented with charcoal-stripped FBS (CH-FBS). Oil using GraphPad Software (GraphPad Inc., CA, USA). Red O (ORO) staining of lipid droplets in differentiated p < 0.05 was considered statistically significant. adipocytes revealed significant inhibition of lipid droplet accumulation in the E treated group compared to that in Results the control group (p < 0.05). Similarly, a significant dose- Optimization of chromatographic conditions dependent inhibition of lipid droplet accumulation was To investigate the content of DYT compounds, 8 active observed in the DYT treatment group (p < 0.001), and the compounds, each indicator component contained in a DYT 400 and 500 μg/mL treatment groups exhibited a combination of herbal medicines, were quantitatively higher lipid droplet accumulation inhibitory effect than analyzed using HPLC/DAD. The active compounds to the E group (p < 0.001) (Fig. 2c, d). In addition, total tri- be analyzed were set based on Korean Pharmaceutical glyceride (TG) and total cholesterol (TC) levels were sig- Affairs. The wavelength for detection was determined by nificantly reduced by E treatment compared with those 2 Jung et al. Applied Biological Chemistry (2023) 66:6 Page 6 of 12 (a)(b) (c) (d) (e) (f) Fig. 2 Eec ff t of DY T on cell viability and anti‑adipogenesis in 3T3‑L1 cells. Cell viability by DY T in 3T3‑L1 cells (a). Data are presented as mean ± SD of four experiments. ** p < 0.01, *** p < 0.001 vs. control. A brief overview of 3T3‑L1 differentiation (b). Optical microscope images of intracellular lipid droplets of differentiated 3T3‑L1 cells (× 200) (c). Graph representing the quantification of lipid accumulation in each treatment group (d). Data are presented as mean ± SD of four experiments. * p < 0.05, *** p < 0.001 vs. control, ### p < 0.001 vs. E . The quantification of total triglyceride ( TG) for each treatment group (e). The quantification of total cholesterol ( TC) for each treatment group (f). The results are presented as the mean ± SD of four independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control., ### p < 0.001 vs. E in the control group (p < 0.001 and p < 0.05, respectively). Inhibitory effect of DYT on the expression of adipogenesis In the DYT treatment group, the TG level showed a sig- regulatory genes nificant decrease in a DYT dose-dependent manner, and Adipocyte-specific transcription factors and regulatory similar to the ORO result, a better decrease was observed genes are closely related to adipocyte differentiation. at DYT 400 and 500 μg/mL than E (p < 0.001). Finally, a To investigate the effects of DYT on the differentiation- significant decrease in TC levels was observed at DYT related genes of 3T3-L1 pre-adipocytes, the expres- 500 μg/mL compared with that in the control group sion levels of PPARγ, C/EBPα, and FABP4 mRNA were (p < 0.001) (Fig. 2e, f ). These results suggest that DYT has analyzed by quantitative real-time PCR (qRT-PCR). As an inhibitory effect on adipocyte differentiation or lipid shown in Fig. 3, PPARγ, C/EBPα, and FABP4 mRNA accumulation in estrogen deficiency and has an effect levels were significantly lower in the E treatment group equal to or greater than that of E in terms of inhibitory than in the control group (p < 0.05). In the DYT treat- efficacy. ment group, the expression of C/EBPα was dramatically reduced at 300 μg/mL concentrations or higher (Fig. 3b), whereas the expression of PPARγ and FABP4 was sig- nificantly reduced at 400 μg/mL concentrations or higher (Fig. 3a, c). Furthermore, compared with the E treatment 2 Jung et al. Applied Biological Chemistry (2023) 66:6 Page 7 of 12 (a) (b) (c) 1.5 1.5 1.5 1.0 1.0 1.0 * * ## ### 0.5 0.5 0.5 *** ### ## *** ### *** *** *** *** 0.0 0.0 0.0 ConE2 200300 400500 ConE2 200 300400 500 ConE2 200300 400500 DYT (g/mL) DYT (g/mL) DYT (g/mL) Fig. 3 Eec ff t of DY T on the expression of adipogenic‑related mRNA in differentiated 3T3‑L1 cells. The relative expression levels of PPARγ (a), C/ EBPα (b), and FABP4 (c) mRNA by qRT‑PCR analysis. The data were normalized using the GAPDH as internal control. The results are presented as the mean ± SD of three independent experiments. * p < 0.05, *** p < 0.001 vs. control., # p < 0.05, ## p < 0.01, ### p < 0.001 vs. E group, PPARγ, C/EBPα, and FABP4 mRNA levels were in the treatment groups treated with 500, 1000, and significantly decreased at 400 and 500 μg/mL. These 1500 mg/kg DYT (Fig. 5c). For HDL-cholesterol, a sig- results suggest that DYT has the potential to improve nificant decrease was observed only in the OVX + HFD lipid production and accumulation due to E reduction group compared to the sham group, whereas a significant and is a mechanism for the inhibition of key factors such increase was observed in the SV and DYT 1500 mg/kg as PPARγ, C/EBPα, and FABP4, which are factors that BW treatment groups (Fig. 5d). regulate adipocyte differentiation. Inhibitory effect of DYT on fat accumulation in liver Weight(s) and serum lipid profile in an animal model and adipose tissue To determine whether DYT (500, 1000, or 1500 mg/kg We further investigated whether DYT inhibits fat accu- BW) treatment could improve post-menopausal symp- mulation in the liver and peritoneal fat tissue. First, as a toms in ovariectomy (OVX), the rats were treated with result of staining with ORO to confirm fat accumulation E , simvastatin (SV), or DYT (Fig. 4a). In the ovariec- in the liver tissue, an increase in fat accumulation was tomized and high-fat diet model experimental groups, observed in the OVX and OVX + HFD groups compared the body weights were significantly elevated com - to the sham group. In particular, an increase in fat accu- pared to the sham group. Weight gain was significantly mulation was observed in the OVX + HFD group com- decreased in the DYT-treated group compared to the pared to that in the OVX group. This fat accumulation OVX + HFD group (Fig. 4b). Uterine weight was signifi - improved in the E and SV treatment groups, and a dose- cantly decreased in all OVX groups (Fig. 4c). The weight dependent decrease in fat accumulation was observed in of intraperitoneal fat was significantly higher in the OVX the DYT treatment group (Fig. 6a). Histological analysis and OVX + HFD groups than in the sham group and of peritoneal adipose tissue showed that the size of adi- lower than that of the OVX + HFD group in the group pose tissue was significantly increased in the OVX + HFD treated with E and DYT1500 (Fig. 4d). Daily food intake treatment group, and the number of adipose tissues was was significantly increased in the OVX group compared significantly decreased. Similar to the liver tissue results, to the sham group and decreased significantly in the significant improvement was confirmed in the E and E and DYT treatment groups compared to the OVX SV treatment groups, and a significant dose-dependent group. For the HFD-fed group, there was a tendency to improvement effect of DYT treatment was also observed increase; however, the difference was not statistically (Fig. 6b). significant (Fig. 4e). The serum levels (s) of TG were also elevated in the OVX and OVX + HFD groups, and The tail‑ or infrared body‑temperature the levels were significantly reduced in the DYT-treated To investigate the anti-hot flash effects of DYT on body group (500, 1000, and 1500 mg/kg BW) (Fig. 5a). In the temperature, we measured the body (tail) or whole-body serum cholesterol profile, TC was significantly increased infrared temperature six weeks after DYT treatment in in the OVX or OVX + HFD group compared to the sham OVX + HFD rats. The tail temperature was elevated in group and was significantly reduced in the DYT 1000 the OVX + HFD group, and its induction was decreased and 1500 mg/kg/BW treatment groups (Fig. 5b). In addi-by E or DYT treatment in OVX + HFD rats. The tail tion, LDL-cholesterol levels were high in the OVX and temperature also fluctuated in the OVX group. However, OVX + HFD groups, and the lowest level was observed this difference was not significant. In addition, changes in c/ebp/gapdh fabp-4/gapdh Jung et al. Applied Biological Chemistry (2023) 66:6 Page 8 of 12 (a) (c) (b) *** *** * ## ### OVX+HFD (e) (d) *** ** ### OVX+HFD Fig. 4 Eec ff t of DY T in OVX or OVX + HFD rat models. A brief overview of the animal model experiments (a). Body weight (b), uterine weight (c), peritoneal fat weight (d), and daily food intake (e). Normal diet group: Sham; ovariectomized normal diet group: OVX; ovariectomized high‑fat diet: OVX + HFD; ovariectomized high‑fat diet plus 17β‑ estradiol 40 μg/kg: OVX + HFD + E ; ovariectomized high‑fat diet plus simvastatin 20 mg/ kg: OVX + HFD + SV; ovariectomized high‑fat diet plus 500 mg/kg DY T: OVX + HFD + DY T500; ovariectomized high‑fat diet plus 1000 mg/kg DY T: OVX + HFD + DY T1000; ovariectomized high‑fat diet plus 1500 mg/kg DY T: OVX + HFD + DY T1500. Data are presented as mean ± SD (n = 7). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sham or OVX group., †p < 0.05 vs. OVX group., # p < 0.05, ## p < 0.01, ### p < 0.001 vs. OVX + HFD group Uterine weight (g) Sham OVX OVX+HFD SV DYT500 DYT1000 DYT1500 Jung et al. Applied Biological Chemistry (2023) 66:6 Page 9 of 12 Triglyceride Total cholesterol (a)(b) *** 150 ** *** ### 100 ### ### ### ### ### ### 0 0 OVX+HFD OVX+HFD (c)(d) HDL-cholesterol ** ## OVX+HFD Fig. 5 Eec ff t of DY T on the serum lipid profile in ovariectomized high‑fat diet animal model. Triglyceride (a), total cholesterol (b), LDL ‑ cholesterol (c), and HDL‑ cholesterol (d) were measured using a competitive enzyme‑linked immunosorbent assay, and data was analyzed using software PRISM (Graphpad; CA, USA). Data are presented as mean ± SD (n = 7). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sham or OVX group., # p < 0.05, ## p < 0.01, ### p < 0.001 vs. OVX + HFD group temperature using infrared thermometers were observed mRNA. We also confirmed the DYT-induced improve - in different patterns in the torso and tail, which were ment in body mass, serum parameters, histological observed only in the OVX + HFD group (Fig. 7). The tail lesions, and clinical symptoms of menopausal obesity temperatures were elevated in the OVX or OVX + HFD in HFD-fed animal models after OVX. In addition, an groups, and its induction was effectively reduced by E improvement in tail skin temperature with weight gain or DYT treatment. However, torso temperature tended was confirmed. These findings imply that DYT has ther - to be decreased in the OVX + HFD group and slightly apeutic potential in the prevention of obesity by reduc- increased by DYT treatment (Fig. 7a, b). ing the weight increase caused by estradiol deficiency. Previous studies have suggested that estrogen defi - ciency can lead to obesity. Due to the local differences Discussion in adipocytes for each sex hormone receptor, estrogen This study aimed to evaluate whether DYT could induces mainly peripheral fat storage via the recep- improve menopausal obesity. In vitro results using tors ERα, whereas androgens contribute to visceral 3T3-L1 cells showed that DYT improved adipocyte dif- abdominal fat accumulation [21, 22]. The decrease in ferentiation, lipid accumulation, and TG and TC levels estrogen following menopause causes a relative andro- through downregulation of PPARγ, C/EBPα, and FABP4 gen excess, leading to a redistribution of body fat mg/dL mg/dL Sham Sham OVX OVX OVX+HFD OVX+HFD E2 E2 SV SV DYT500 DYT500 DYT1000 DYT1000 DYT1500 DYT1500 mg/dL Sham OVX OVX+HFD SV DYT500 DYT1000 DYT1500 Jung et al. Applied Biological Chemistry (2023) 66:6 Page 10 of 12 (a) (b) *** *** * ** *** *** # 1500 ### ### ### ### ### ### ### ### ### OVX+HFD OVX+HFD Fig. 6 Eec ff t of DY T on fat accumulation in liver and adipose tissue. The accumulation of lipid droplets in the liver tissue was evaluated by ORO staining (× 400), and ORO‑positive areas were analyzed using ImageJ software (a). The size and number of adipocytes in the adipose tissue were evaluated by H&E staining (× 400), and the size and number of adipocytes were analyzed using ImageJ software (b). Data are presented as the mean ± SD (n = 7). *p < 0.05, **p < 0.01, ***p < 0.001 vs. sham or OVX group, #p < 0.05, ###p < 0.001 vs. OVX + HFD group (a) (b) Fig. 7 Eec ff t of DY T on the tail‑ or body‑ temperature. Tail skin and infrared body temperatures were evaluated with an infrared thermometer or thermography, respectively (a), and data of tail‑skin temperature was analyzed using software PRISM (Graphpad; CA, USA) (b). Data are presented as mean ± SD (n = 7). ***p < 0.001 vs. Sham group or OVX group. #p < 0.05, ##p < 0.01 vs. OVX + HFD group (mainly visceral abdominal fat) [22–24]. In addition, In this study, body weight, daily food intake, perito- an increase in all-trans-retinol 13,14-reductase (RET- neal fat weight, TG, TC, LDL-C, and HDL-C levels SAT), which promotes adipogenesis in adipocytes, and were improved by DYT in ovariectomized HFD-fed adipose tissue lipoprotein lipase (AT-LPL), which is animal model. In particular, weight gain increased in involved in FFA production, absorption, and storage, the OVX + HFD group compared to that in the OVX were reported in a study in OVX mice [25–28]. Owing group, but there was no significant change in the to these effects, the increased FFA produced from the daily food intake, confirming that estrogen decrease excessively accumulated visceral abdominal fat flows due to ovariectomy was the main cause (Fig. 4b and into the liver and increases insulin resistance [29]. Fur- e). In addition, uterine weight decreased in the ovar- thermore, decreased estrogen synthesis due to ovarian ian removal group. This suggests that DYT does not dysfunction increases CVD prevalence by increasing induce endocrine disturbances (Fig. 4c). In addition, LDL-C, a substrate for E , and decreasing HDL-C [27]. histological analysis of adipose tissue confirmed that Adipocytes area (m ) Sham OVX OVX+HFD Tail skin temperature (°C) E2 SV Sham DYT500 DYT1000 OVX DYT1500 OVX+HFD Number of adipocytes perfield E2 Sham DYT1500 OVX OVX+HFD E2 SV DYT500 DYT1000 DYT1500 Jung et al. Applied Biological Chemistry (2023) 66:6 Page 11 of 12 Funding DYT decreased the area of peritoneal adipocytes and This research was funded by the Korea Institute of Oriental Medicine (Grant increased the number of adipocytes. Similarly, inhibi- Nos. KSN 1515290 and KSN20224314). tion of lipid accumulation in the liver tissue by DYT Availability of data and materials was confirmed. The datasets used and/or analyzed during the current study available from the PPARγ plays a key role in adipocyte differentiation and corresponding author on reasonable request. maturation [30]. Activated C/EBPα by C/EBPβ and C/ EBPδ, which are expressed at the early stage of adipocyte Declarations differentiation, induce mutual expression with PPARγ Competing interests and cooperate with the activation of adipocyte genes The authors declare that they have no competing interests. such as FAS and FABP4 [7]. In premenopausal women, PPARγ activity is downregulated by estrogen-activated Received: 26 September 2022 Accepted: 19 December 2022 ERα, and estrogen loss with menopause increases the effect of PPARγ to promote weight gain [31]. Therefore, we induced 3T3-L1 differentiation by treating DYT in a phenol-free medium containing CH-FBS to block hor- References monal interference (Fig. 2b). It was confirmed that adi - 1. 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Journal
Applied Biological Chemistry – Springer Journals
Published: Jan 27, 2023
Keywords: Danggui-yukhwang-tang (當歸六黃湯, Dāngguīliùhuángtāng); Menopausal syndrome; Adipogenic differentiation; Obesity